Isolation And Identification Of A Bacterial Strain Capable Of β-Aminopropionitrile Bioconversion Into β-Alanine And Biotransformation Process In Bioreactors

As a multipurpose organic material for preparation of carnosine and pantothenic acid（Vitamin B₅）which itself is a component of coenzyme A,β-alanine plays an important role in fine chemical and pharmaceutical synthesis.The main content of this research are as follows:Isolation and identification of a bacterial strain G20 capable ofβ-aminopropionitrile bioconversion intoβ-alanine;Optimization of biotransformation process forβ-alanine production by resting cells in a bubble column reactor,and by immobilized cells in a highly compact multi-phase-fluid reactor and a packed-bed reactor,respectively,after investigating on preparation and characterization of the immobilized cells.Based on the characteristics of morphology,physiology and biochemical tests,Biolog GP2 identification,and 16S rDNA sequence analysis,strain G20 was identified as Rhodococcus erythropolis.From the preliminary research on biotransformation process,we tentatively put forward two possible mechanisms for convertingβ-aminopropionitrile toβ-alanine by Rhodococcus sp.G20.One possibility:The strain of Rhodococcus sp.converted nitrile through a two-step reaction,catalyzed by a nitrile hydratase-amidase system.The other possibility:The strain of Rhodococcus sp.harbored nitrilase,nitrile hydratase and amidase,and the initial formation ofβ-alanine was partly attributed to nitrilase.A 2³ central composite experimental design was performed with the purpose of optimizing theβ-alanine production by resting cells in a bubble column reactor with 200 mL working volume using response surface methodology（RSM）.The optimum bioconversion conditions in a batch operation were as follows:cells loading of 16.50 g_ww/200 mL,substrate concentration of 1.29%（v/v）,and airflow rate of 86.56 L/h,with the corresponding productivity ofβ-alanine 6.33 g/（L.h）.The influences of the temperature and initial pH of a buffer solution on the conversion were also studied,and the optimums were 30 ^C and pH 7.5.The measured activation energy（E_a）was found to be 22,199 J/mol,thus indicating the presence of diffusional resistance.The biocatalyst’s half-life(t_1/2)was found to be 46.83h,which corresponds to almost 11 batches used in a bubble column reactor.Application of Ca-alginate-entrapped cells of Rhodococcus sp.G20 treated with polyethyleneimine（PEI）and glutaraldehyde（GA）in turn for improving mechanical strength inβ-alanine production has been studied. The optimum conditions for immobilization obtained by orthogonal test were as follows:cells concentration of 5%,sodium alginate concentration of 3%,CaCl₂ concentration of 3%,and bead diameter of 2 mm. Furthermore,the individual and interactive effects of PEI concentration,pH, and Ca²⁺concentration on the capability of bioconversion and mechanical strength of Ca-alginate immobilized cells were investigated by RSM. Maximum%conversion was achieved when PEI concentration,pH,and Ca²⁺concentration were set as 0.34%,8.0,and 41.75mmol/L,respectively, whereas,optimal mechanical strength was achieved when the variables above were set as 0.48%,7.6,and 52.15mmol/L,respectively. The research on the characteristic of immobilized cells showed that the optimal results were achieved in the biotransformation system of saline, with substrate concentration of 1.2%（v/v）,at 35℃.The half-life of this biocatalyst treated with PEI and GA was found to be 12 batches.Application of a highly compact multi-phase-fluid reactor inβ-alanine production by immobilized cells revealed an obvious drop in activity after 3 batches indicating that introduction of air would cause damage to immobilized cells.Biotransformation process in a packed-bed reactor was also investigated.The half-life of this biocatalyst was roughly 3 days in continuous operation with the optimal conditions:preincubation time 15h at 40℃and feeding speed of 0.2 mL/min at 35℃.